Turkish Journal of Veterinary and Animal Sciences
Turk J Vet Anim Sci (2015) 39: 634-636 © TÜBİTAK doi:10.3906/vet-1504-81
http://journals.tubitak.gov.tr/veterinary/
Short Communication
Prevalence of blaZ gene and other virulence genes in penicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases in Gansu, China 1,
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Feng YANG *, Qi WANG , Xurong WANG , Ling WANG , Min XIAO , Xinpu LI , 1 1 1 Jinyin LUO , Shidong ZHANG , Hongsheng LI 1 Key Lab of New Animal Drug Project, Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Science, Lanzhou, Gansu, P.R. China 2 State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu, P.R. China Received: 23.04.2015
Accepted/Published Online: 21.07.2015
Printed: 30.10.2015
Abstract: Staphylococcus aureus is a major etiological agent of bovine mastitis worldwide. In this study, 37 strains of S. aureus resistant to penicillin were isolated from bovine mastitis cases in Gansu Province for investigating blaZ and virulence-related genes, including tst, eta, etb, lukPV, lukED, lukM, hla, hlb, hld, and edin. Antibiotic resistance was based on disk diffusion method and blaZ and virulenceassociated genes were studied by polymerase chain reaction. Penicillin resistance gene blaZ was detected in 35/37 (94.6%) of penicillinresistant S. aureus isolates. tst, lukPV, lukED, hla, hlb, and hld were observed in 5.4%, 2.7%, 89.2%, 70.3%, 73.0%, and 70.3% of the penicillin-resistant isolates, respectively, while eta, etb, lukM, and edin were not detected in any isolates. blaZ carried by penicillinresistant S. aureus isolates may be the main reason for phenotypic penicillin resistance. Virulence determinants encoded by lukED, hla, hlb, and hld genes may play important roles in bovine mastitis pathogenesis of penicillin-resistant S. aureus in Gansu Province. Key words: Bovine mastitis, Staphylococcus aureus, antibiotic resistance, blaZ, virulence-related genes
Staphylococcus aureus is the primary contagious pathogen in bovine mastitis, causing severe economic losses to the dairy industry worldwide (1). Penicillin is the most commonly used drug in the treatment of mastitis (2), which has led to an increase in the number of resistant strains (3). The pathogenic basis of S. aureus and its response to antibiotic therapy depend on various antibiotic resistanceand virulence-associated genes carried by the pathogen (4). Resistance gene blaZ is responsible for resistance to penicillin (5). Currently, over 40 virulence-associated genes have been reported among various S. aureus strains (6). Although some but not all of the virulence-related genes in S. aureus strains from bovine mastitis have been reported in South and East China (7,8), little is known yet about the virulence genes in penicillin-resistant S. aureus strains recovered from bovine mastitis in Northwest China. Therefore, the aims of this work were first to determine the genetic basis of penicillin resistance and second to investigate virulence-related genes in penicillinresistant S. aureus strains from bovine mastitis cases in Gansu Province. * Correspondence:
[email protected]
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Thirty-seven bacterial isolates from bovine mastitis (26 isolates from clinical cases and 11 isolates from subclinical cases) were collected at 3 farms located in Gansu Province in China during 2014. Mastitis infection was confirmed by the California Mastitis Test. The isolates were identified as penicillin-resistant S. aureus strains by morphological characterization, biochemical testing, and disk diffusion method according to Clinical Laboratory Standards Institute standards (9). S. aureus ATCC 29213 was used as control isolate. The penicillin-resistant gene blaZ and several virulence-related genes encoding toxic shock syndrome (tst), exfoliatins (eta, etb), leukotoxins (lukPV, lukED, lukM), hemolysins (hla, hlb, hld), and EDIN (edin) were detected by simplex PCR according to Olsen et al. (10) and Jarraud et al. (11), respectively. As shown in the Table, 94.6% of S. aureus isolates resistant to penicillin were shown to have the expected penicillin resistance gene blaZ. lukED was the most prevalent virulence gene (89.2%), followed by hlb (73.0%), hla (70.3%) hld (70.3%), tst (5.4%), and lukPV (2.7%). eta, etb, lukM, and edin were not detected in any strains. In the blaZ-positive isolates, lukED, hla, hlb, and hld were the most commonly occurring virulence-related genes,
YANG et al. / Turk J Vet Anim Sci Table. Distribution of blaZ and virulence-related genes in penicillin-resistant S. aureus from cows with mastitis in Gansu. Resistance genotype
No. (%)
No. (%) of virulence-related genes
of strains
tst
eta
etb
lukPV
lukED
lukM
hla
hlb
hld
edin
blaZ
35 (94.6)
2 (5.4)
0 (0)
0 (0)
1 (2.7)
32 (86.5)
0 (0)
25 (67.6)
26 (70.3)
25 (67.6)
0 (0)
blaZ-
2 (5.4)
0 (0)
0 (0)
0 (0)
0 (0)
1 (2.7)
0 (0)
1 (2.7)
1 (2.7)
1 (2.7)
0 (0)
Total
37 (100)
2 (5.4)
0 (0)
0 (0)
1 (2.7)
33 (89.2)
0 (0)
26 (70.3)
27 (73.0)
26 (70.3)
0 (0)
+
blaZ +: Strains positive for blaZ; blaZ -: Strains negative for blaZ.
detected in 86.5%, 67.6%, 70.3%, and 67.6% of isolates, respectively. In contrast, tst and lukPV were found in only two isolates and one isolate, respectively. For the two blaZnegative S. aureus isolates, the detection rates of lukED, hla, hlb, and hld were all 2.7%. The activated blaZ could encode β-lactamase enzyme (penicillinase), which inactivates the antibiotic through hydrolysis of the peptide bond in the β-lactam ring (5). In this study, not all the penicillin-resistant S. aureus isolates exhibited genotypic resistance to penicillin, corresponding well to the findings of previous studies (12). This is in agreement with data from Gao et al. (13), showing that no resistance genes could be determined in some phenotypically resistant Streptococcus agalactiae isolates. In some isolates, phenotypic resistance may have been caused by point mutations rather than gene acquisition. Additionally, except for the general resistance mechanisms (14), other pathways such as biofilm formation may play a major role in the resistance mechanisms (15). The penicillin-resistant S. aureus isolates in the present study showed a high frequency of lukED. The lukED gene could encode bicomponent leukotoxins with which S. aureus can target and kill innate immune cells critical for defense against bacterial infections (16). A high prevalence of hla, hlb, and hld was also observed in the S. aureus isolates, in agreement with findings reported in China and other countries (7,17–19). Hemolysins encoded by hla, hlb, or hld aid the S. aureus population in
the invasion of the host cells and cause damage (20). The observation of frequent lukED, hla, hlb, and hld suggest that bicomponent leukotoxins and hemolysins encoded by these corresponding virulence-related genes may have crucial roles in pathogenesis of bovine mastitis caused by penicillin-resistant S. aureus in Gansu Province. In conclusion, our results suggest that the blaZ carried by penicillin-resistant S. aureus may play a major role in the penicillin-resistant phenotype, but the resistance gene cannot be used alone as a diagnostic indicator for penicillin resistance. Further studies should be performed to develop accurate molecular indicators of antibiotic resistance. In addition, the detection of genes encoding virulence determinants suggests that lukED, hla, hlb, and hld are the most prevalent virulence-related genes in penicillin-resistant S. aureus from bovine mastitis cases in Gansu. These results emphasize the need for monitoring the genetic basis of antimicrobial resistance and virulence determinants in S. aureus. Acknowledgments This study was supported by the Special Fund of the Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (No. 1610322015007), the National Key Science and Technology Support Project in the 12th Five-Year Plan of China (No. 2012BAD12B03), and the Natural Science Foundation of Gansu Province (No. 145RJYA311).
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